TARGETING TOLL-LIKE RECEPTOR 4 WITH COMPUTATIONAL CHEMISTRY METHODS AND RATIONALLY DESIGNED GLYCOCONJUGATES

Author

• Olivia Slater, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2026

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Kontoyianni, Maria

Abstract

Toll-like Receptor 4 (TLR4) and protein partners lymphocyte antigen 96 (MD-2), Cluster of Differentiation factor 14 (CD14), and High Mobility Group Box 1 protein (HMGB1) are immune receptors involved in inflammation. TLR4 Agonists include bacterial lipopolysaccharide (LPS), ganglioside GM3, and chemotherapy drug paclitaxel. Earlier work employed a structure-based design strategy to develop potent amino acid monosaccharides (AM) that bind with TLR4/MD-2 at the protein-protein (PP) interface to antagonize LPS. Compounds 12, 101, and 104 had IC50s equal to 470 nM, 13 nM, and 311 nM, respectively, and 101 was biphasic. Taking into consideration these prior findings, research was undertaken to address questions pertaining to the cause of the biphasic response, chemical features that are important for activity at TLR4/MD-2, feasibility of small molecule design for these receptors, GM3 targeting with glycoconjugates, and whether the power of computational modeling could identify putative binding partners of paclitaxel. These overarching questions established the basis toward the project’s objectives. Rational design, ab initio calculations, docking, and machine learning were employed in order to assess the selectivity of AM conjugates and explore structure-activity relationships. Results show that 101 binds with TLR4/MD-2 and MD-2 alone, charge and polarity on activity and selectivity are significant, and acetyl protection of the amino acid is the best chemical reduction. Computational predictions were confirmed experimentally. Molecular dynamics, PP docking, and virtual screening were undertaken in order to assess the viability of the PP interfaces of TLR4 and partners, and to investigate the feasibility of small molecule binders of CD14. Results show that interfaces between TLRs and one pocket on CD14 are amenable to molecular design. Finally, de novo design, enhanced sampling, and free energy calculations were carried out in order to design glycoconjugates and elucidate paclitaxel’s likely binding partners in metastatic disease. A robust protocol was developed, and results show that a designed glycoconjugate behaves differently in metastatic versus healthy bilayers, and that HMGB1 with TLR4/MD-2 are putative binding partners of paclitaxel.